JPH07503139A - How to use branched nucleic acid probes - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention]

METHODS OF USE OF BRANCHED NUCLEIC ACID PROBE FIELD OF THE INVENTION The present invention relates generally to nucleic acid probes designed for use in hybridization assays. BACKGROUND OF THE INVENTION Nucleic acids are formed from nucleotide bases such as uracil (U), antidine (C), adenine (A), thymine (T) and guanine (G) and are formed as single-stranded linear molecules. Such a linear molecule can be connected to another linear molecule, for example, A and T, A,! : By forming specific base pairs between U or G and C, hybrid complexes or double-stranded components are formed. molecules (also called duplexes) can be formed. Comparing such paired molecules It is called a complementary molecule. Nucleic acid hybridization is a method in which two single-stranded complementary molecules form a double-stranded molecule. This method is typically used to detect the presence of single-stranded molecules in a sample (target nucleic acid) through the use of labeled probes formed from complementary single-stranded molecules. For example, 1iuran et al. [1087104165 (published July 16, 1987)] described a single-stranded region complementary to the nucleic acid to be detected and a non-radioactive label. A probe having a double-stranded region with a bell is disclosed. Conventional use and design of nucleic acid probes is well known in the art, and the present invention relates to novel nucleic acid probes and methods of their use. The present invention provides an alternative method that can form a detectable structure only in the presence of a target nucleic acid. is based on the use of further probes. Usually at least two of the nucleic acid molecules portion (which may be on a different molecule) hybridizes with the target nucleic acid to generate this structure. You have to create a structure. That is, this probe ensures that few or no false positive results are observed. Such probes can be used to detect nucleic acids and can also be used as therapeutic agents. These allow new detection methods to be performed independently of the structure of the target nucleic acid. Furthermore, amplification of the label signal is easily achieved and thus these probes allow very high detection sensitivity of target nucleic acids. Therefore, in a first aspect, the present invention provides for at least two separate target-specific regions and at least two distinct regions that do not hybridize to the target nucleic acid but retain complementary regions that can hybridize to each other. The present invention relates to a nucleic acid hybridization probe having at least one nucleic acid strand having a genomic region. Under appropriate hybridization conditions, complementary arm regions will not hybridize to each other in the absence of target nucleic acid, but will hybridize to each other in the presence of target nucleic acid. The target-specific region of the arm anneals to the target nucleic acid, and the complementary arm regions anneal to each other. These regions are designed to anneal to thereby form a branched nucleic acid structure. In a related aspect, the invention relates to methods of using such probes to detect the presence or amount of a target nucleic acid in a sample. In this method, a predetermined One or more nucleic acid molecules are provided that together contain at least two independent regions that hybridize to a target nucleic acid under controlled ambient conditions. These molecules also contain at least two arm regions that do not hybridize (under predetermined ambient conditions) to the target nucleic acid or to each other in the absence of the target nucleic acid. However, the target nucleus In the presence of acid, the arm regions hybridize to each other. This method further When a target nucleic acid is present, there is a preservative that allows hybridization of the arm region. contacting the sample with the nucleic acid molecule under ambient conditions determined for the purpose of the test. Any hybridization of the arm region is then detected as an indication of the presence or absence of the target nucleic acid. In a preferred embodiment, the detection step involves contacting the arm region with a solbase (this term refers to an enzyme capable of degrading a linkage point, such as a 4-way linkage or a 3-way linkage, by linkage-specific cleavage) and 1 or more nucleic acid molecules Detection of cleavage by Rubase or DNA footprint of further nucleic acid molecules observing the mobility of one or more nucleic acid molecules within a gel matrix, detecting the binding of an intercalator to one or more nucleic acid molecules, 81 Nuclease contact and nucleases detection of any cleavage of l or more nucleic acid molecules by l or more enzymes; contacting the above nucleic acid molecule with a restriction endonuclease and detecting cleavage of another nucleic acid molecule by the endonuclease, or measuring the thermal stability of one or more nucleic acid molecules. In another preferred embodiment, the or more nucleic acid molecules include an insertion molecule or a nucleic acid binding molecule (e.g., an acridinium ester), or another molecule (where the molecule is a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule). It is susceptible to chemical or other modification by acids or bases if it forms part of a single-stranded nucleic acid molecule or or otherwise insensitive to such chemical cleavage when present in a double-stranded nucleic acid molecule. the detection step includes one or more nucleic acid molecules and a chemical involves measuring the quality of contact and the amount of chemical modification of a molecule, in which at least one of the one or more nucleic acid molecules has 8 to 30 (or 8 ~100) has a target region containing complementary bases; In the absence of the target nucleic acid, the region of the genome is hybridized under predetermined ambient conditions. form a duplex with a melting temperature (usually referred to as Tm and measured as explained below) that is 4°C, most preferably 7°C or even 10°C below the reaction temperature, and in the presence of the target nucleic acid. have a TID at least 4°C (or 7°C or even 10°C) above the hybridization temperature under predetermined ambient conditions. form a double strand. In yet another preferred embodiment, there is provided a nucleic acid molecule having a loop region connecting at least two arm regions: 1 or more. One or more nucleic acid molecules consist of two nucleic acid molecules, each having a target region and an arm region; one or more nucleic acid molecules consist of three nucleic acid molecules, each of which has a target region and an arm region; each having at least one arm region, and at least two nucleic acid molecules having independent target regions, wherein the three nucleic acid molecules are hyperlinked to the target nucleic acid. hybridizes to form at least two independent hybridized or double-armed regions. one or more nucleic acid molecules consisting of four nucleic acid molecules, each of which has at least one arm region, and one or more nucleic acid molecules, each of which has at least one arm region; The four nucleic acid molecules and the target nucleic acid are redize to form at least three independent duplexes between the arm regions. In yet another preferred embodiment, the target region hybridizes to the target nucleic acid and The arm regions hybridize together to form an arm such that a point of connection is formed at the base of the arm between two separate target regions. one or more nucleic acid components The child or target nucleic acid may include an arm region or a target region or a nucleic acid near the point of attachment that does not form a duplex with the target nucleic acid but loops out from the point of attachment. Also, the sign The target regions are located along their entire length or at their distal ends away from the arm regions. It includes nucleic acids that do not form duplexes with acids and therefore either loop from the duplex formed between the target nucleic acid and the target region or extend as a single-stranded region from the end of the target region. In yet another option, the arm region may include a loop that extends from the arm region without forming a duplex with other arm regions, or a single-stranded molecule from the end of the arm region away from the target region. This includes nucleic acids that extend as for one example In this case, the target regions hybridize together to form arms, forming two independent targets. A connection point is formed at the base of the arm between the target areas. One or both of the arm regions can hybridize to the other arm region at the end furthest from the target region. It further has a single-stranded region that is flawed and thus available for duplex formation with another nucleic acid molecule to form a second arm. In this example, one or more nucleic acid components The child forms a duplex with the single-stranded region to attach the second or third arm and the second attachment point. It may include a portion that may be formed between the arms. In a related preferred embodiment, at least two arm regions are in contact with the target nucleic acid. When redized, it forms an arm that contains biologically or chemically active sites, such as restriction endonuclease sites, double-stranded regions, and single-stranded regions (where double-stranded regions are DNA polymerase act as a primer for RNA polymerase), a promoter for RNA polymerase, e.g. DNA-RNA duplexes susceptible to cleavage by RNA5eH, and substances chemically active against cleavage of adjacent duplex nucleic acids, such as Fe-EDTA. Additionally, the arm or target region may include a single-stranded region that can be looped across the double-stranded region to form a double-stranded nucleic acid. Such triple-stranded structures are well known in the art and can be easily designed as part of the present invention. Wear. Additionally, such single-stranded nucleic acids can be provided independently of the rest of the nucleic acid molecules and can be labeled by conventional methods, if desired. Also, this can also be used as an arm. may even form part of the target region. Detection of double-stranded nucleic acids is readily accomplished by methods known in the art. contacting other nucleic acid molecules capable of hybridizing with the regions or single-stranded regions extending from these arm regions to form one or more double-stranded regions or arms; further hybridize the nucleic acid molecules between them. It can be increased to form multiple arms or arm regions. Therefore, the probe described above is incubated with a sample suspected of containing the target nucleic acid under appropriate hybridization conditions to detect the hybridization of the complementary arm region. Detection of hybridization can be used as an indication of the presence of target nucleic acid. Typically, probes are two or more independent nucleic acid molecules, e.g. formed from long chains between groups: detection of acridinium ester within the tract Monitor the presence of ibridase 7-J. In another related aspect, the invention provides a method for creating a biologically or chemically significant site that may be remote from a target nucleic acid but is formed only in the presence of the target nucleic acid. related. This site is relatively inactive with respect to a particular chemical or biochemical activity when in single-stranded form, but becomes active with respect to a particular chemical or biochemical activity when hybridized with its complementary strand. or vice versa. It is a component. Examples of chemical or biochemical activities include duplex-specific chemical cleavage. (e.g. using lron-EDTA?1 conjugation or phenanthyl lolin to duplex nucleic acids to create duplexes sensitive to copper cleavage), restriction enzymes Donuclease digestion, RNA5eH digestion (even if one strand of the duplex is RNA) The other is a site sensitive to DNA), or a site that can be acted upon by T7 RNA polymerase, or a site capable of DNA polymerase activity, such as primer extension activity. (e.g., using Taq DNA polymerase). It is not limited to these. Other examples include digestion when in the form of single-stranded nucleic acids (by Sl nuclease). This includes sites that are protected from such digestion when the protein is in double-stranded form. Nucleic acid hybridization probes containing this site are Such a site can be designed as described above, except that it includes a probe, or it can be created when the probe hybridizes to a complementary arm region. Therefore, this professional the target nucleic acid to form a branched nucleic acid structure as well as the desired site. Ru. Additionally, chemical or biochemical actions are performed as desired. This can be used, for example, as a mode of nucleic acid detection, a mode of amplification, or a mode of target-mediated cleavage. Wear. In yet another related aspect, the invention provides methods for mediating biological activity in target cells by hybridizing probes to specific target sites, as described above. Regarding the law. As described above, the probe is incubated with the target nucleic acid to generate complementary conjugates. A branched nucleic acid structure is formed that includes a double-stranded region formed between the double-stranded and double-stranded regions. Examples of target nucleic acids in this method include essential sequences within the genome of a pathogenic organism; essential mRNA sequences produced by cells and essential sequences in cancer cells (essential means that the viability of an organism or cell in the absence of such sequences is determined by methods well known in the art). (meaning a reduction of at least 30%). plow The double-stranded region formed between the complementary arm regions of the antibody is a sequence-specific antibody recognition site. (specific binding antibodies may mediate therapy by a variety of well-known mechanisms, e.g., a cytotoxin attached to the antibody would kill the cell) and sequence-specific protein/enzyme (Treatment may be mediated by enzymatic cleavage, blocking of transcription, blocking of translation, etc.) designed as a specific recognition site for one of various therapeutic approaches, including It is measured. Additionally, the points of attachment between nucleic acid strands can serve as recognition sites for such antibodies or proteins. Other features and advantages of the invention will be apparent from the following description of preferred embodiments of the invention and from the claims. Description of preferred embodiments 4.

[Brief explanation of the drawing]

Layer Planes Figures IA-IF, 4A-4B, 6A-6H, 9A-9F and IIA-IIF are schematic diagrammatic representations of the probes of the present invention (the short lines between the probes in each figure are Figures 2A-2E and 3A-3B, 5A-5G, 7 and 8.10 and 12A-12C are FIGS. 13 and 14 are specific examples of mismatched probes; FIGS. 15A-15F are specific examples of probes shown in FIGS. A diagrammatic representation of various detection systems that are useful: Figure 16 This is a specific example of a probe that can be detected by donuclease treatment. Figure 17 is an autoradiogram of a test using the probe shown in Figure 16; Figure 18 is a specific example of a probe detectable by enzymatic amplification; and 19B1 and 20A-20C are schematic representations of target-independent probe amplification systems. FIG. 21 is a diagrammatic representation of three probes creating a four-way connection point; and FIGS. 2A-22+ are diagrammatic representations of various embodiments of the probes shown in FIG. Ru. Probes Referring to Figures IA and IB, one example of a probe of the invention is shown, which illustrates the general structure of each probe of the invention. The probe shown is a hybrid It is formed from two independent strands 10.12, each carrying a target-specific region 14.16 that is capable of hybridizing to a target nucleic acid 15 under isostatic conditions. Also, each chain Arrays that normally hybridize together to form arms 22 only in the presence of a target nucleic acid. 18.20. When the arms are formed in this way, a connection point 21 between the arms and the hybridized target region is also formed. The arm region and the target-specific region are hybridize to target nucleic acids and to each other in their arm regions. Hybridization of the target-specific region to the target nucleic acid is called hybridization. can be designed to be observed under observation conditions. Accordingly, as generally explained above, the nucleic acid hybridase-7 molecules of the present invention The probe creates a branched nucleic acid structure (i.e., extends directly from the target nucleic acid) by interacting with and hybridizing to the target nucleic acid. a structure having at least one arm that does not bridle). The probe is designed so that the formation of this branched structure is target-dependent. Detection of branch formation is thus a measurement of the presence and amount (if detection or quantitative) of a target nucleic acid in a sample. One or more branches can be formed, but only one branch is must be formed in a dependent manner. The probe can be a single stranded nucleic acid or wL several strands. Additionally, the strand (or strands) may be DNA, modified DNA5RNA, modified RNA, or various combinations thereof. “Modified DNA or RNA refers to any nucleic acid structure that differs from naturally occurring DNA or RNA molecules, such as modified bases (e.g. syn) or modified internucleotide linkages (e.g., phosphorothioate or methyl phosphonate). The probe is the target nucleus at least two independent regions that hybridize specifically with the acid, and at least two regions that are complementary to each other and form a stable duplex only in the presence of the target. must have a hybridizing region (i.e., a region that does not hybridize with the target). No. Because these complementary arm regions hybridize only in the presence of the target nucleic acid, this jetting of duplex regions is the preferred method for assaying for the presence of the target nucleic acid. There are numerous methods for detecting hybridization of branched nucleic acid probes and targets. Examples of detection of branched nucleic acids include junction-specific cleavage with lysyl-based enzymes (e.g., to detect cleavage of labeled probe strands), DNA5e footprinting, gel retardation assays, and labeled junction-specific insertion compounds. binding (target is captured) the presence of the label is detected). Although purified four-way linkage point-truncated lysyl bases are well known in the art, three-way linkage point-cleaving enzymes and other linkage point-specific enzymes can be used, e.g., to cleave the desired three-way linkage point. It can be easily purified by biochemically purifying an enzyme that exhibits cleavage activity. An example of detecting duplexes formed between complementary arm regions includes the DNA5e fragment. 31 nuclease digestion (labeling the arm region: not digested by nuclease S1 if the arm forms a duplex; Restriction endonuclease cleavage of the site created by duplex formation between the complementary arm regions (which would otherwise be digested by nuclease S1), thermal stabilization of the bound duplex following capture of the target nucleic acid. analysis (using labeled probe strands: complete) A fully branched nucleic acid structure will have higher thermal stability than any partial formation and will If no branching occurs, a different signal will be associated with the captured target nucleic acid than if a branch was formed. Also, the juxtaposition of two collaborative labels on opposing probe arms can be detected. More generally, one skilled in the art will readily recognize that any method that detects the formation of branched DNA or complementary arm duplex formation can be used in this method to indicate the presence of the target. . A preferred method of detection is the use of the hybridization protection assay (HPA) as disclosed by Arnold et al. [35C11n, Chem, 1588 (1989)]. This method uses a chemiluminescent acridinium ester (AE) label covalently attached to a synthetic oligonucleotide probe. This assay format is completely homogeneous (i.e., does not require any physical separation steps) and is based on chemical hydrolysis of the ester bond of the AE molecule, the cleavage of which permanently renders the AE non-chemiluminescent. do. Hydrolysis of this ester bond is fast for unhybridized AE-labeled probes (AE-probes), but for hybridized Slower conditions have been developed for AE-probes that have been moved. Therefore, after hybridization of the AE-probe and its complementary nucleic acid (under conditions that do not promote ester hydrolysis), reaction conditions can be adjusted to remove unhybridized AE-probe. The chemiluminescence associated with hybridized AE-propylene rapidly decreases to low levels, but Chemiluminescence associated with the lobes should be minimally affected. This differential hydration After the decoupling step, any residual chemiluminescence is a direct measure of the amount of duplexes formed. Ru. or more AE labels to one or more nucleic acid strands of the probe. and assayed for duplex formation using the differential hydrolysis method described above. Therefore, this assay method is used in the present invention. Two-component Probes One arrangement of branched nucleic acid probes used for detection of target nucleic acids is shown schematically in Figures IA and IB. In this configuration, the probe consists of two independent nucleic acid strands. These strands are preferably synthetic oligonucleotides. Each strand connects to the target nucleic acid. It holds the probe region and arm region that hybridize. The two arm regions are complementary to each other (this means that they are not completely complementary to each other). This does not necessarily mean that they are perfectly complementary, as they may have some conjugation, but it does mean that a stable duplex will be formed under hybridization conditions). The melting temperature, Tm, of the bound duplex in the absence of target is adjusted so that little or no hybridization of the arm regions occurs in the absence of target nucleic acid. lower than the operating temperature, preferably 4°C (more preferably 7°C or 10'C) These arm regions are designed so that The Tm is defined as the temperature at which 50% of a given nucleic acid duplex has melted (ie, become -channeled). Tm depends on ambient conditions such as the cation concentration of the solution. The desired Tm is typically achieved by manipulation of the length and nucleotide base composition of the complementary regions. Also, incorporation of mismatches, substitution of inosine for some or all of the guanosine residues, e.g. Other methods can be used to adjust the Tm of the duplex, including, but not limited to, modification of the phosphate backbone with sphorothioate internucleotide linkages. HPA type When the formula is used at 6°C, the preferred length of the fully complementary, unmodified arm region is about 8-20 contiguous bases (depending on base composition and sequence). other surrounding strips conditions may lead to a range of different magnitudes; this is easily measured empirically. It will be done. Upon contacting the probe with a solution containing the target nucleic acid, the probe regions of the two strands will hybridize to their respective target regions, usually in close proximity to each other (they are directly connected to each other), as shown in Figure 1. They do not need to be in contact, see below). this When this occurs, the arm regions of the two probe strands are forced into close proximity to each other, thus increasing the stability forces of the bound duplex. arms form a double strand so that the Tm of the duplex formed in the presence of target is approximately equivalent or The arm region is designed to be above the operating temperature of the assay, preferably 4°C (more preferably 7°C or 10°C) above the operating temperature. As indicated above, when using the HPA format at 60°C, the preferred length of the arm is about 8 to 2 o contiguous complementary bases (depending on base composition and sequence). Two independent stranded probe regions can be designed in a variety of ways. For example, these regions can be designed similarly to the arm regions, but in this case the Tm of either region alone (i.e., one probe strand + target strand) is lower than the operating temperature. However, when both probe strands and target strands are present and the arm regions hybridize, the temperature is higher than the operating temperature. Also, the Tm of both probe regions is higher than the operating temperature. They can be designed such that one Tl is higher than the operating temperature and one Tm is lower. The design is selected Whatever is chosen, the requirement that the arm regions form a stable duplex only in the presence of the target must be met. The probe region is preferably 8-5o nucleotides. The length is preferably between 8 and 30 nucleotides, more preferably between 8 and 30 nucleotides in length. These regions may be longer, but this additional length is not necessary for most applications. Furthermore, the synthesis of these longer oligomers is more expensive and time consuming than shorter oligomers. One or both probe sequences are reacted with the desired target nucleic acid, preferably any The target nucleic acid is selected so that it does not react (ie, cross-react) with undesired target nucleic acids. If one probe region hybridizes to the undesired target while the other probe region hybridizes to the undesired target, If not, both probe sets are required for the arm region to hybridize. The assay will still function properly since the components must hybridize. When using the HPΔ format, it is common to place the AE label in the arm region of the probe. In the absence of target, this region will be single-stranded and the AE will be susceptible to rapid ester hydrolysis. In the presence of target, this region will become double-stranded and the AE will undergo V4XJ from ester hydrolysis. AE labels can be placed in one strand, the other strand, or both channels (this allows for signal and and lead to increased assay sensitivity). Multiple labels can be placed within each tract, which further improves assay sensitivity. Also, place the AE label on one side of the probe area. or the other (or both). Also, multiple labels could be used in this arrangement. Furthermore, any set of arrangements described above AE could be used in combination. To detect target nucleic acids in a sample using the branched nucleic acid probes described above in the HPA format, the following general method is used. 1) Add the branched nucleic acid probe to the sample, 2) Incubate to cause annealing of the appropriate regions, and 3) Add the branched nucleic acid probe to the sample. Add drug to perform differential hydrolysis of the AE label, and 4) remove any markers present. Any residual chemiluminescence is detected as an indication of the presence or amount of target nucleic acid. Annie Ring conditions can be varied depending on the exact application, the design of the probe, the nature of the target nucleic acid, and the composition of the sample containing the target. However, the above Tm is required. Conditions must be selected to meet the requirements. I-I P A format For this purpose, the incubation temperature is preferably between 5 and 70°C, more preferably between 30 and 65°C, and the pH of the annealing step is preferably between 4.5 and 8.5, most preferably between 4. .5 to 5.5 (although higher stability of AE during the annealing step is achieved at relatively low pH ranges, Hammond et al. [6J, Biolum, and exist) . The concentration of the -valent salt counterion is preferably between 50 and 1000 mM, more preferably Preferably between 200 and 800mM. This prevents the AE from agglomerating and sticking to the walls of test tubes and pipette tips), preferably at a concentration of 0.1-10% (w/v), more preferably 1-10%. is preferred. Alternative Annealins Leading to Desired Hybridization Characteristics One of ordinary skill in the art will appreciate that conditions that can be used in the present invention may also be used in the present invention. Specific examples are provided below, but are not a limitation of the invention. Many modifications possible An example is provided in Figure IC-IF, where various loops (labeled L) are shown in the arms, target nucleic acids or junctions, as will be understood by those skilled in the art. Waterway nucleic acids (labeled S) may also be present (e.g., (for later duplex functions) with more probes such as A specific example of the general arrangement shown in FIG. 1 is shown in FIG. The target strand is a synthetic NA oligomer 43 bases in length with a sequence corresponding to the region of the 23S liposomal RNA (rRNA) subunit of Mycob acterium tuberculosis. The two strands of the branched nucleic acid probe are the target strand and the target strand, as shown in Figure 2A. Together they form a three-way connection point. As shown, one AE is covalently linked between the T and A residues of chain 1. Oligonucleotides were synthesized on a Biosearch Model 8750 or ABh 3 80^ DNA synthesizer using conventional phosphoramidite solid-phase chemistry [Caruther et al. Using polyacrylamide gel electrophoresis and purified. Chain 1 was converted into an AE label as previously disclosed by Arnold and Nelson [PCT Publication No. 108 9102896 (published April 6, 1989)]. It rang. The following annealing reaction was carried out in 30 μl of O, 1M lithium succinate buffer (pH 5). 2), 2mM EDTA, 2mM EGTA and 10% (w/v) lithium lauryl sulfate at 60'C for 1 hour. Hybrid-target strand (0,5p moiety) ), probe strand 1 (AE larabelled strand) (0,0!1 mol) and probe strand 2 (2,5 p mol). Control 1 - probe strand 1 (0,05 pmol) and probe strand 2 (2,5 pmol). Control 2 - Probe strand 1 (0.05 pmol). reagent bran - No target or probe. To show that the arm region hybridizes only in the presence of target, The half-life of AE hydrolysis for each reaction described above was determined using a previously disclosed protocol [Arnold et al., 35 C11n Chem, 1588 (19g9)]. The following results were obtained: Control 1: 054 min Control 2: 051 min These data indicate that either AE or target is not protected from ester hydrolysis in the absence of (Compare Control 2) was shown to be protected from hydrolysis in the presence of the target (hybrid), thereby indicating that the arm region is protected from hydrolysis only in the presence of the target nucleic acid. It shows that Ibridize is the best. This is done by measuring the Tm of various configurations, which are represented schematically in Figure 2B. The system was further evaluated. Tm was measured according to the following protocol. 1. After annealing as above, the sample to be analyzed was diluted in 0.1M lithium phosphate buffer (pH 5,2), 2mM EDTA, 2mM EGTA, 10% (W/V) lithium lauryl sulfate to 100 μm. Approximately 100,000 phases per Relative light units (RLU; this is the chemiluminescent unit used in these protocols; see Arnold et al. E35 C11n Chcm-1588 (1989)). 2. Pipette 100 μm aliquots into 12×75+am assay tubes. Each sample to be analyzed was run for 7 minutes at each of the following 4 degrees: 56°C and below for oligomers with a Tm of 40, 43.46.49.52.55. Incubate at 58 and 62°C and 50.53.56.59.62.65.68 and 71°C for oricomers with Tm higher than 56°C. Ta. After incubation, each test tube was placed in a water bath. 3. After all incubations are complete, remove all test tubes from the water bath and 300 μl of 0.15 M sodium tetraborate buffer (pH 7.6) and 5% (V/v) Triton X-100 were added to each well and mixed by vortexing. All tubes were incubated at 40°C for 20 minutes for oligomers with Tm below 56°C and for 15 minutes at 50°C for oligomers with Ta above 56°C. The test tube was placed on ice for 30 seconds and then placed at room temperature (20-25°C). 4. Chemiluminescence was measured in a luminometer (LEADERl, Gen-Probe, CA) by automatic injection of 20 μm of 0°1% H,O,,1N NaOH, followed by 5 s. It was measured by measuring the signal between. Before measuring chemiluminescence, place the test tube in a damp container. Plotted with a cloth or paper towel. 5. Subtract the control value from each corresponding hybrid value and use the resulting data for chemical development. Plotted as light versus temperature. Let Tm be the temperature at which 50% of maximum chemiluminescence is lost. This was determined based on the graph. The temperature range and exact temperature interval used can vary with the exact nature of the AE-oligomer being characterized. The following Tms were obtained: Structure 1 (Figure 28) Ta+ = 62°C Structure 2 (Figure 20) Tm = 50° °C (hydration (the operating temperature used above in the kinetics measurements) and a Tl11 higher than 60°C in the presence of target. The various structures of Ta shown in Figures 2B-2E were prepared in 8mM lithium succinate buffer (pH 5,5), 100mM NaCl, 10mM MgCL and 10+aM Tris-HCI (this reagent was the dilution reagent in step 1 of the above protocol). Niyo This system was further characterized by measurements in the 2000 µm (2000 µm). The following T m were obtained: Structure 1 (Figure 28) Tm = 59°C Structure 2 (Figure 2C) Tm = 47°C Structure 3 (Figure 2D) Tm = 47°C Structure 4 (Figure 2E) Tm = 45 For the individual designs at °, not only is the Tm of the arm region much lower in the absence of target than in the presence of target, but the duplex region formed between the target and probe regions is a complete arm duplex. more stable in the absence of a fully-armed duplex (see Figure 2) than in the presence of strands. Qualitativeness is low. This indicates that the branched nucleic acids of the present invention can be used to manipulate the Tm of nucleic acid duplexes. Other possible arrangements for the acridinium ester label position or the use of restriction enzymes for duplex detection are shown in Figures 2B-2E. (Hereinafter, blank spaces) Example 2 Another example of the general structure shown in FIG. 1 is shown in FIGS. 3A and 3B. In this design, all oligomers were synthesized as described in Example above, where liposomal RNA (rRNA) derived from Nelsseria gonorrhoeae was the target nucleic acid. and AE-labeled. Two basic designs (99/135 Figure 3B design and +32/146 Figure 3A design) with junction points at slightly different positions within specific regions of the rRNA were evaluated. Various linker-arm configurations were evaluated as shown below. In addition, a precisely complementary target nucleic acid (N, gonorrhoeae) as well as a latent with two mismatches Currently cross-reactive target nucleic acids (N, meningiLidis) were evaluated. Using differential hydrolysis and Tm analysis as described in Example 1, the following specific conditions were measured: The hybridization properties of different regions were evaluated in the following cases: I N,gon, 0.6 pmol AEo, 1 lmol None 2pmol none 2pmol 2 N,gon, 0.6 pmol None 2pmol AE(1 ) 0.1 p mol None 2 p mol 3 N, gon, 086 p mol AEO, lp mol AE (1) 0.1 p mol None 2 p mol 4 N, gon, 0.6 p mol None 2 p mol^E ( 2) 0.1 pmol none 2 pmol5 N, men, 0.6 pmol to E O,l pmol none 2 pmol none 2 pmol6 N, rnen, 0.6 pmol Le None 2pmol AE (1) 0.1 No Pmol 2pmorph N, men, 0.6 pmol ^E0.1pmol AE (1) 0.1 No pmol 2p mol9 N, gon, 0.61 ) Mol A20.1 pmol None 2 pmol None 2 pmol 10 N, gon, 0.6 pmol None 2 pmol AE 0. 1 pmol None 22 moles 11 N, gon, 0.6 pmol AEo, 1 pmol Le AE O,l ppmole to 2pmol12 N, men, 0.6 pmol to E0.1pmol none 2pmol none 2pmol13 N, men, 0.6 pmol none 2pmol AE(1) O,l No pmol 2 pmol N, hybridization with N. gonorrhoeae was performed at 60°C; hybridization with N. meningitidis was performed at 45 or 50°C; controls included all components except target: Linear oligonucleotides 100 and 146 are "helper" probes that open the second structure of the rRNA and are the branched nuclei of the present invention. Not related to acid probes. The following results were obtained. 1 N, gon 6B, 2 14.3 0.48 29.82 N, gon 7 0.0 6.5 0.51 12.73 N, gon 68.0 8.2 0.50 16.44 N, gon 68.5 18.8 0.47 40.09 N, gon 70.0 12.8 0.49 26.110 N, gon 72. 0 8.0 0.43 18.6II N, gon 67.5 8.6 0.43 20.05 N, men 59.0 6 Nll1en 59.0 7 N, men 54.5 8 N, men 56.0 12 N, men 58.5 13 N, men 61.0 14 N, men 57. The O DH ratio is the ratio of half-lives of hybrid, de and control. Nelsseria meningiLidis DH ratio is close to unity (data not shown). Therefore, ha Iburitaisenyon was not observed. The DH ratio to N, gonorrhoeae target clearly shows that the arm region forms a stable duplex only in the presence of the RNA nucleic acid target. Each of these two designs has a strand with a long probe region and a strand with a short probe region, demonstrating the flexibility of this design parameter. Furthermore, the data show that several different AE configurations, including 2 AEs per probe (one on each strand; samples 3 and 11), produce similar results. Some AE configurations yield relatively higher DH ratios than others, but all exhibit protection only in the presence of a target. vinegar. N, gonorrh. The Tm data for the eae target show that all structures tested yield Tms significantly higher than the operating temperature (60° C. in this case). Data obtained by N. Meningitidisli indicate that the system can be tailored to not significantly cross-react with well-correlated target nucleic acids, thereby improving its utility as an assay. . The Tl11 differences for each structure between N. golo rrhoeae and N. meningitidis range from 92 to 13.5 °C. The 99/135 system was also evaluated for its ability to detect reduced amounts of target nucleic acids (N, gonorrhoeae) and cross-reactivity with 10-' μg (63 fmol) of N. meningitidis was also tested. The assay format was as follows: target and probe were added to 100 μl of 0.1 M lithium succinate buffer (pH 5,2), 2 mM EDTA, 2 mM EGTA and 10% b/V). Annealing was performed at 60° C. for 60 minutes in lithium uryl sulfate. 300 μl of 0.15 M sodium borate buffer pH 7.6) containing 5% (v/v) hlydon X-100 detergent was added, vortexed and incubated at 60° C. for 10 minutes. Chemiluminescence using a luminometer (LEADERI, Gen-Probe, C^) Measurements were made by automatic injection of 200 μm of 0.4 N HNO3, 0.1% H2○2, then 200 μl of IN NaOH, followed by measurement of the signal for 5 seconds. In this example, the following probe mixture was used. Probe mixture 1 - AE-labeled oligo 99 (0,1 p mol), oligo l 35 (0,5 p mol) and oligo 146 (2 p mol) Nibrope mixture 2 AE-labeled - kl) probe mixture 3-AE-labeled oligo 99 (01 pmol), AE-labeled oligo 135 (0,1 pmol), oligo 99 (0,:)l) mol) and oligo 146 (2 pmol), ) and Oligo 146 (2 pmol). 1 of the targets assayed and the probe mixtures for which the results are shown below. These data demonstrate that the design presented here is useful in the detection of small amounts of nucleic acids in a complete assay format. Furthermore, label amplification is shown in probe mixture 3 containing two A E-labeled strands instead of one (as in probe mixtures 1 and 2). Non Cross-reactivity with N. meningitidis RNA, which is always well correlated, is minimal for probe mixtures 1 and 3; The slight cross-reactivity obtained was about 100 times lower than the positive signal, indicating that A. It was reduced to a minimum level by slightly increasing the operating temperature of the cell. Single-Component Probes Another arrangement of branched nucleic acid probes used for detection of target nucleic acids is schematically shown in Figures 4A and 4B. In this configuration the probe consists of a single stranded nucleic acid. desired characteristics The properties are the same as described for the design shown in Figure 1, but now the two strands of the probe are joined, thus becoming one strand. The segment 24 joining the two halves of the probe may be nucleotide or non-nucleotide in nature and should be of a length to provide the desired probe properties described above. When using the HPA format at 60°C, the preferred length of the complementary, unmodified arms 25.27 in this configuration is due to the intramolecular nature of the interaction and is similar to the design shown in Figure 1. It is slightly shorter than the one mentioned above. Therefore, the preferred arm length is about 4 to 16 bases. Ru. As mentioned above, other ambient conditions may lead to a different size range, which is easily determined experimentally by routine methods. Example 3 An example of the general structure shown in FIG. 4 is shown in FIG. The target strand is a 43 base long synthetic NA oligomer with a sequence corresponding to the region of the 23S rRNA subunit of Mycobacterium a+ tuberculosis. As described in Example 1 above. All oligomers were synthesized and AE-labeled as described above. As shown in Figure 5A. Similarly, branched nucleic acid probes form a three-way junction with the target strand. One AE indicates As shown, the T and G residues of the probe are linked by a co-parenting bond. In addition, the AE was placed in the target strand at one of the two positions shown in the specific analysis as described below. The DH and Tm analysis protocols described in Example 1 were used to evaluate the hybridization properties of the structures shown in Figures 5B-5G; The amounts of each component used are listed in Figures 5B-5G. The following results were obtained: 2 10.2 0.32 31.9 8.1 0.56 14.5 These data indicate that the This shows that the resulting duplex is more stable in the presence of target than in its absence. The relative difference in stability is large enough to be used to unambiguously detect the presence of the target nucleic acid, but not as large as that seen in either Example 1 or 2. Comparing the hydrolysis rates of samples 1.2 and 3 controls, it is apparent that the arms interact somewhat under the operating conditions of this experiment. This interaction can be essentially eliminated by optimizing the operating conditions (eg, higher operating temperatures, inosine residues contained in the arm region or phosphorothioate linkages contained in the arm region). This increases the discrimination between when the target is present and when the mark is absent. Alternative configurations of branched nucleic acid probes used to detect target nucleic acids are shown schematically in Figures 6A-6 (in these configurations, the probes can contain three or more nucleic acid strands). These strands form one or more nucleic acid junctions with the target nucleic acid. As described above, such structures can be engineered into one nucleic acid molecule. The key is that the arm regions form stable duplexes only in the presence of target (the possibility of some arm regions forming stable duplexes even in the absence of target is discussed below). Virtually any combination of connection points is possible. The guidelines for designing such a system are essentially the same as those described above for the arrangement shown in FIG. In the case of a four-way junction (or higher order junction), some of the probe strands will contain only arm regions and no target-specific regions. Since the sequence of the strand containing only the arm region is independent of the target sequence, it can be used in combination with various specific probe strands for the detection of various target sites. This “universal detection” app Roach offers several advantages, including very few (or even one) AE-labeled universal detection oligomers for detection of all target sequences (which significantly reduces design and synthesis time). This includes the ability to use (minimizing the amount of hydrolysis and therefore minimizing cost) and the freedom to choose sequences that yield optimal differential hydrolysis, complete independence from the target sequence. As mentioned above, AEs can be placed in some or all arm regions as well as some or all probe regions. One advantage of this arrangement is against AE. The presence of many sites for the label amplification of the assay Significantly increase. In one particular use, the sequence around the AE site can be the same for each arm region, which determines the AE hydrolysis properties (AE hydrolysis rate). (the accuracy is sequence-dependent) and improves the overall accuracy of the assay. Professional The length of the tube region can be manipulated to achieve certain assay performance goals. For example, by adjusting the length of various probe segments, cross-reactivity with highly correlated targets can be minimized. For example, Figures 6A, 6B and and 6C, the probe regions 30 and 32 of probe strands l and 3 are relatively long; Therefore, it forms stable duplexes with targets and even highly related non-target nucleic acids that retain homologous sequences in these regions. The probe region 34 of probe strand 2 is relatively short and spans a region containing l or more mismatches 36 to the non-target nucleic acid. This probe segment will form a stable duplex with a perfectly matched target, but due to its relative shortness it will not form a stable duplex with a mismatched target. The example above shows a three-way connection between an arm and two target areas. As shown in Figures 6E and 6F, three probes (which may be formed as one molecule) with independent arm regions (labeled A) and probe regions (P) are used. can create a four-way connection point. Similar examples are shown in Figures 68 and 6G. Example 4 A specific example of the general structure shown in FIG. 6A is shown in FIG. The probes are labeled 99 strands, 135 strands and 146 strands. As in Example 2, liposomal RNA (rRNA) from Neisgeria gon orrhoeae is the target nucleic acid. All oricomers were synthesized and AE-labeled as described in Example 1 above. Contains three probe strands together that form two three-way nucleic acid junctions with the target strand. Two basic designs were evaluated. The only difference between the two designs is the inclusion of a Roubouara) (L) base at the linkage point (optional) as shown in Figure 7. Many loop bases can be contained in the probes of the invention, which are non-null nucleotide bases, and may also form probes that create non-immobilized or mobile attachment points). Various linker arm configurations were evaluated as shown below. In addition, exactly complementary target nucleic acids (N, gonorrhoeae) as well as 2 mismatching potentials The cross-reactive target nucleic acids (N, N. meningitidis) were evaluated. Using differential hydrolysis and Tm analysis as described in Example 1, different regions of the hive Redization properties were evaluated under the following specific conditions: Structures 1 to 18 are shown schematically in Figure 8: target strand (0.6 pmol), AE-labeled probe strand (0.1 pmol) and unlabeled probe strand In samples to -12, the target was N, meningitidis; in all other samples, the target was N, gonorrhoeae; in samples 1, 2.4 to 6.1 0. As shown in FIG. 7, ILs 13, 14, 16, and 17 had a roubou out base, and the remaining samples did not have a lubou out base. The obtained data are shown in tabular form in FIG. These data indicate that multiple arm regions can be formed in the presence of target, and only in the presence of target. Furthermore, multiple AE labels can be used in one design. This can lead to label amplification. Additionally, these structures can be formed with and without the Luboout base shown in FIG. Additionally, selected structures from those shown in Figure 8 were evaluated for their ability to detect the target nucleic acid (N, gono rrhoeae): Cross-reactivity with N, meningitidis was also tested. The general assay format was the same as that used in Example 3, with the following probe amounts: 2 AE-labeled strands-01 pmol; unlabeled probe ta-2p mole. Le. The amounts of target nucleic acids assayed and the results are shown below: Structure Non-target (see Figure 8) lo-2 μg N, gon, 10-2 μg N, men vs. These data demonstrate that the design presented here is useful in the detection of small amounts of nucleic acids in a complete assay format. show. Additionally, significant label amplification occurs in structure 16. This is an alternative to one (as in probe structures 1.4 and 5). Each contains three AE-labeled strands. Cross-reactivity with the highly correlated N,’men ingitidis RNA was minimal for structure 16, and the assay By slightly increasing the operating temperature of the pond (chosen as optimal for structure 16), some cross-reactivity seen in the pond structure can be reduced to minimal levels and used for the detection of target nucleic acids. Another arrangement of branched nucleic acid probes is shown schematically in Figures 9A-9F. This arrangement is very similar to the structures just mentioned, where the probe consists of three or more nucleic acid strands, such as those shown as 40, 42, 44, which have two or more nucleic acid joining points. form, but this If one or more junctions are formed with the target nucleic acid (e.g., 1 in Figure 9A) and l or more junctions do not bind to the target (e.g., junction 2 in Figure 9A) still formed only in the presence of. The arm regions form a stable duplex only in the presence of target (they form a stable duplex even in the absence of target). (The possibility of several arm regions forming the Any combination is possible. Some examples of structures having this property are shown schematically in Figures 9A-9F. The guidelines for designing such systems are essentially the same as for the configurations described above. The best combination of lengths and sequences for each region of each chain is determined experimentally by testing various structures until the optimal combination is achieved. It is normal for this to occur. The variations and advantages of this arrangement are shown in the arrangement represented by FIG. This is essentially the same as explained above. Example 5 A specific example of the general structure shown in FIG. 9 is shown in FIG. The target is a synthetic NA 60mer (T) with sequences corresponding to major genetic translocations associated with chronic myeloid leukemia. (See Example 6 below). The probe consists of three strands, two of which form a three-way linkage with the target, and one of them forms a three-way linkage with the other two. This third strand has only one arm region and is itself a possible universal detection oricomer as described above. Although one AE labeling site is detailed in this example, AEs can be placed in other areas as well. To evaluate the capabilities of this system, DH and Tm analyzes were performed as described in Example 1 with the following specific conditions: target strand - 05 pmol; unlabeled probe strand = 2 pmol. Differential hydrolysis was performed at 506C. The results are shown below. These data are clearly due to the lack of protection of the AE-labeled strands against AE hydrolysis. As shown, the two junctions of this structure form only in the presence of target (at operating temperature). This is a unique situation in which hybridization of strands that do not even make contact with the target strand is completely target dependent. This is the universal detection oligomer described above. clearly demonstrate the concept of It is also possible to detect chimeric nucleic acid targets using branched nucleic acid probes in stratified configurations that form multiple junctions in response to the target. A chimeric nucleic acid consists of two or more segments, each of which originates from a different source or is otherwise unique in some way (e.g., the chimeric DNA is present only in a portion of a population of organisms). It is a nucleic acid consisting of An example of such a chimeric target is the product of a gene translocation shown schematically in FIG. In this case, one chromosome segment is translocated onto another chromosome (and vice versa in reciprocal translocations), creating a chimeric DNA molecule (which may or may not result in a chimeric mRNA). do. This type of translocation can occur in multiple regions. can occur with a cut point (on one or both chromosomes), resulting in A plurality of chimeric DNAs are obtained. FIG. 11B represents the case where three independent breakpoints on chromosome 2 are recombined with one breakpoint on chromosome 1. This type of chimeric target can be detected in many ways, examples of which are shown in Figures 11C-11F. Method 1 (Figure 11C) is a sandwich method that requires a physical separation step. This is a test assay. This method uses a capture probe specific to chromosome 1 (this capture probe The probe is labeled with a specific capture substance C, which allows the probe to be specifically removed from the solution. For example, C may be biotin; The heterogeneous capture support may be avidin agarose), and the three of chromosome 2 Three independent detection oligomers (each with a reporter group) specific for different translocation regions of (labeled). Method 2 (Figure 11D) utilizes AE-labeled probes specific for each translocation product. (This is a homogeneous assay in which the breakpoint junctions are (This is done by designing a Method 3 (FIGS. 11E and 11F) utilizes the method of the present invention and involves branching nuclei. An acid structure is formed around the breakpoint connection point (other structures are possible in this application, such as those shown in the previous figure). Chain a is universal for all translocation products, and chains d, f and h are specific for the corresponding translocation products. In this specific example In , strand a is the only strand that is AE-labeled. This system has several advantages, including: It is homogeneous, which is not required in method I. Requires no physical separation step: Has a universal sequence for AE protection, leading to uniform differential hydrolysis properties for each target: Has only one AE-labeled strand; Reduces background with minimal sloppiness and cost; less sensitive than method 2 to incorrect pairings (which can occur in the breakpoint junction region) (See Example 7). The guidelines for designing such a system are essentially the same as for the arrangement described in Figure 1 above. Example 6 A specific example of the general arrangement shown in FIG. 11 is shown in FIGS. 12A-12C. Chimera target staining It is a synthetic NA oligomer (80mer) homologous to three different gene translocations between the constant ABL region of chromosome 9 and the variable region of chromosome 22. Two are the most common translocations associated with chronic myeloid leukemia (CML) and one with acute lymphocytic leukemia. disease (ALL). Each chimeric target possesses the same abl region, but all regions derived from chromosome 22 are different. In addition, the normal abl gene (separated on both sides) An 80mer corresponding to the 4o base (cross-linking point 4o base) was synthesized. One AE-labeled strand specific for the abl region was designed as shown in Figures 12A-12c. If the probe region is specific to one of the 22 translocated chromosome regions, Three different chains were designed to contain arm regions complementary to those of the universal detection oligomer. We first wanted to evaluate the ability of these three designs by measuring the differential hydrolysis rate and Tm for each using the protofur described in Example 1. Target-0° 5 pmol; AE-labeled probe fa-0 , 1 pmol; unlabeled flow chain -2 pmol). The results are shown below 1 18.3 0.636 28.8 71.52 18.4 0.640 2 8.8 -m-318,60,64029,1--1 Next, the ability of the three-probe mixture to detect suitable target sequences (and not react with inappropriate target sequences) was evaluated. The assay format described in Example 4 was used (20 fmol of target; 0.1 pmol of AE-labeled probe strand, 2 Pmol of non-Lachelated probe strand). The results are shown below (average of 4 reactions). Lobe Probe AE-Label Mixture 1 Mixture 2 Mixture 3 Chain Mi1 202.187 1,354 1.265 8B92 2.950 16 7.614 1,343 1,1073 1.307 2,319 131.6 70 729 Normal abl 1.491 1,248 1.094 886 Target None 624 614 506 451 The probe mixture detects only the correct chimeric target and does not significantly intersect with either the other two chimeric targets or the normal abl sequence. There is no differential reaction, thereby demonstrating the utility of this arrangement for detecting chimeric nucleic acid targets and The use of a single AE-labeled probe strand to detect multiple targets is shown. Mismatch Probes In another arrangement, different targets with related but non-identical sequences are detected using branched nucleic acid probes. Typically, DNA probe-based assays are designed to detect a specific target and not cross-react with highly correlated targets. Indeed, considerable effort has gone into developing assays that would be sensitive to as few as one mismatch. I feel strong. However, the assay is relatively insensitive to mismatches. There are cases where it is necessary to An example of such a case is the detection of viruses such as HIV that exhibit significant genomic changes. Example 7 A specific example of the above general structure is shown in FIG. The precise target strand is a 78 base long synthetic NA oligomer with a sequence corresponding to the region of the gag gene in HIV-1. Another synthetic 78 IIler was also constructed to contain various mismatches as shown in Figure 13 (these sequences correspond to various HIV chains that have been isolated and sequenced). Two branched nucleic acid structures (see Figure 14) with identical probe regions but different arm regions were evaluated. Very stable at operating temperature The probe region was designed to be unique, thus increasing the overall stability of the branched nucleic acid structure once formed with the target. added by the arm region (hybridization The stability of each region, including the stability (if acts as a buffer against loss of stability in the region, thus reducing the susceptibility of the entire structure to mispairing. All oligomers were prepared as described in Example 1 above. was synthesized and labeled with AE. The differential hydrolysis ratio and Tm of these designs were calculated using the general protocol shown in Example 1 above. Measurement was carried out using a fool under the following specific conditions. Target - o, sp mol; AE-labeled strand - 0.1 p mol; non-uberized probe strand - 2 p mol; hybridized probe strand - 2 p mol; The temperature and corresponding DH ratio analyzes were carried out at three different temperatures (see below 60, 55 or 50°C). The results are as follows. 1 1 1 060 4.3 0.56 7.7 -2 10.2 0.82 12.4 -1&2 7.4 0.75 9.8 -1 1 0 eo 5.9 0.77 7.7 952 3 7.7 0.77 10 593 4 6.1 0.77 7.8 6546 10.0 0.7713 6 1 1 055 +6.5 1.1 15 B52 3 13.9 1.1 1 2.6 793 4 13.6 1.1 12.4 854 6 9.8 1.1 8.9 571 1 0 50 30.3 1,7 17.8 912 3 41.6 1.7 24.5 763 4 32.2 1.7 18.9 8 74 6 17.8 1,7 10.5 82To+('C) 2 + 1 0 60 10.3 0.87 11.8 742 9.8 1.5 6.5 71 1&2 9 .9 0.58 17 702 and 1&2 1 0 60 9.3 0.66 14.12 3 9.6 0.66 14.6 3 4 9.5 0.86'14.4 4 6 7.2 0. 66 10.9 *-Number of invalid pairs. 10-D Hm degrees (°C). 9-Independent experiment. These results demonstrate that branched nucleic acid probes can tolerate 3 and 4 mismatches with few or no effects on the performance profile, and 6 mismatches with only a small decrease in the performance profile. Therefore, branched nucleic acid probes can be used in assay formats that require relative insensitivity to mispairing. Furthermore, these data again include multiple AEs per probe design. (i.e., label amplification). Structure 2 was further evaluated by detecting the reduced amount of perfectly paired target (Target 1) in the full format assay as described in Example 4 (strands 1 and 2 AE label). (Target concentrations are shown below). The results are as follows. These data demonstrate that the branched nucleic acid probes presented here can detect small amounts of target nucleic acids in a complete format assay. Detection of branched duplexes Target-dependent formation of duplexes between complementary arm regions of branched nucleic acid probes can be detected using methods other than the A E-label/differential hydrolysis method described above. can. Furthermore, this differential duplex formation produces a wide variety of target-dependent biological or chemical properties. can be made to stay the same. Examples of some of these properties, including alternative modes of detection, will be discussed using the double-stranded nucleic acid probe described in Figure 1 as a model. However, it is likely that the arm region in question forms a stable duplex only in the presence of the target. As long as these properties meet the basic criteria of It will be readily appreciated that it can be applied to any tube arrangement. Example 8 Restriction Endonuclease Cleavage In one system (FIG. 15A), arm region duplex formation creates an active (double-stranded) restriction enzyme cleavage site (R) that is removed from the target strand. Cleavage at this site by restriction endonucleases can be detected in a variety of ways. For example, one or both strands can be labeled with 3 marks and the cleavage products can be detected using gel electrophoresis or other separation methods. Ru. Alternatively, one or both strands can be labeled with a capture substance (such as biotin) on one side of the cleavage site and the other side of the cleavage site with a reporter group (such as biotin). For example, the strands can be labeled with “P, AE). After cleavage, the strands can be captured and labeled with e.g. vizin agarose), the reporter group bound to the capture support will not exhibit any cleavage. However, the reporter group bound to the supernatant shows cleavage. Alternatively, the detection probe can be designed to react with the uncleaved strand but not with the cleaved strand. This can be done in a homogeneous or separate assay (a homogeneous format is shown in Figure 15B). In one specific aspect of this system, the cleavage site is designed to be close to the base of the arm region duplex (i.e., at the point of attachment). Upon cleavage by lyase, the arm region is almost completely removed from the three-way split nucleic acid structure, thus reducing the stability of the overall structure. probe area or none Cleavage may occur if the wound arm duplex is designed to be stable only in the presence of the duplex. If this happens, the probe region will melt away from the target. This then hybridizes the uncleaved probe strand to the target and starts the process again. In this way, multiple probes can be circulated through a single target site. This increases the sensitivity of the assay. (Hereinafter, blank space) Example 9 DNA/RNA polymerase elongation In another system (Figure 150), poly The arm region is designed to create a site for elongation by the enzyme. Relatively short chains first 3! This filled region can be detected by P-labeling and then detecting any extended primer strands using gel electrophoresis or other separation methods. You can do something like that. in a differential hydrolysis format (1. AE-labeled protein specific for the extension product). Lobes can be used to detect packed arrays. When restriction sites are contained near the base of the arm region duplex, this system also has potential for circulation as described above. Example 10 Amplification In another system (Figure 15D), loading with polymerase amplifies active T7 RNA protein. Create a lomotor part. Following filling, T7 RNA polymerase will transcribe multiple copies of the template strand, which will result in detectable product amplification and thus increased assay sensitivity. These strands can be used, for example, as guides for radiolabeled ribonucleotides. by separation (e.g. by precipitation) or by complementary AE-labeled proteins. It can be detected by differential hydrolysis of the lobes. When a restriction site is contained near the base of the arm region duplex, this system also has some potential for circulation as described above. RNA transcripts are detected using, for example, AE-labeled probes. Example 11 Selective decomposition In the autopsy system (Figure 15E), one arm region is DNA and the other arm region is DNA. The region (or part thereof) is RNA. Formation of the arm duplex creates an active RNA5e H cleavage site. Cleavage by RNA5eH can be detected as described above in system 1 (Figure 15F). This system also has the potential for cycling as described above. ), a chemical cleaving substance (X, e.g. For example, Fe-EDTA or Cu-phenanthroline) is attached to the arm region of one of the probe strands. If the arm region duplex forms, cleavage of the immediately adjacent nucleic acid occurs (the type of cleavage depends on the chemical cleavage agent and the exact location and attachment chemistry). ). This disconnection can be detected as described above. This system also has the possibility of circulation as described above. Any other system for distinguishing between single- and double-stranded nucleic acids, such as specific monoclonal antibodies against double-stranded nucleic acids, is potentially applicable to the configuration generally described above. It is Noh. Example 13 A specific example of the system shown in FIG. 15A is shown in FIG. This structure does not include the AE label. This is the same as shown in FIG. 2, except that BstEII restriction endonucleo Engineer the ase site into the indicated arm region. To analyze cleavage of the target nucleic acid on the arm region, strand 2 of the probe is subjected to a conventional kinasing reaction. First labeled with 3MP using rotor. The probe was then hybridized to the target using the following conditions: hybrid: 1P-labeled strand 2 (0.6 pmol); If (15 pmol); target strand (3 pmol). Control: Same as Ibrid except for the target. As a control for enzymatic activity, the NA target strand was evaluated as being exactly complementary to the two strands. These controls will be referred to as linear-hybrid and linear-hybrid controls. The amounts used were the same as above (C═P-labeled strand 2 (0.6 pmol): target strand (3 pmol)). These annealing reactions were incubated in 60 μl of 50 mM Tris buffer i& (pH 7,9; at 25°C), 500+ nM NaCl for 60 min at 60°C. I bet. A portion of each annealing reaction was then digested with BstEll under the following conditions. 10 μl of each of the above reactions were diluted to 100 μl with final conditions of 50 mM Tris buffer (pH 7,9; at 25°C), 100 mM NaCl, 10a+M MgCl, 1 mM TT. Supported with either 10.5.1 or 02 units of Bst E I l (New England Biolabs) Samples were incubated at 60°C for 60 minutes. Next, a portion of the reaction solution was poured into a regular pot. It was analyzed using lyacrylamide gel electrophoresis (20% Kel, 500V, 15+nA, 25 hours). A regular autoradiogram of the gel was then performed. The results are shown in Figure 17, showing target-dependent cleavage of arm region duplexes with 10 and 5 units of BstEII. Furthermore, is the arm region sequence independent of the target sequence? The restriction site may be any desired site. This style smell Therefore, the use of restriction enzymes does not depend on finding the correct recognition sequence in the target to be analyzed. It would be nice. A specific example of the system shown in FIGS. 15B and 15G is shown in FIG. This structure contains a template strand (strand 1) and a primer strand (strand 2). The template strand contains a detection sequence at its 5' end and a T7 promoter site 3' of the detection sequence (see Figure 18). (see). Upon hybridization of both mirrors to the target, the arm regions form a stable duplex, and the 3' end of strand 2 provides an active primer extension for the polymerase enzyme. rank. The enzyme loads the complement into the template strand, which is then denatured to form the structure shown in Figure 18. detected using an AE-labeled detection oligomer. Also, polymer Fill-in with enzyme creates an active (double-stranded) T7 promoter region with the same detection sequence template region. The T7 RNA polymerase enzyme then synthesizes multiple RNA copies. can be transferred from the template and is also detected using an AE-labeled detection oligomer. Probes were prepared using strand 1 (0,3 pmol), strand 2 (0,9 pmol) and target strand (0,1 pmol) in 50 mM Tris buffer (pH 7,9; at 25 °C), 0.5 M NaCl Incubate at 60°C for 60 minutes (final trial M10ttl). The control sample that you want to hybridize with the target strand first by won). This sample was then diluted to 50 μm and the DNA polymerase was added under the following conditions. Extended with limerase: 20mM Tris mild solution (at pH 7,9, 256C), 0.15M NaCl, 3mM MgSO, 1% TX-100, 1mM DTT, 2mM dATP, 2OM dTTP, 2mM dGTP. 2mM dCTP and 4 units of Taq Bolimerase (Cetus Corp.) The reaction was incubated at 60°C for 1 hour. A 5 μm equivalent volume of sample was then mixed with 25 μl of 6% lithium lauryl sulfate, 60 mM sodium phosphate buffer (pH 6,8), 2 mM EDTA and 2 mM EGTA and 20 μm water. The sample was incubated at 95°C for 5 minutes, cooled to 60°C or below, and then added with 50 μl of 200 mM lithium succinate buffer (pH 5,2), 17% lithium lauryl sulfate, 2 mM EDTA, 2+ nM EGTA and and AE-labeled detection oligomer (0.1 pmol). This reaction mixture The samples were incubated at 60°C for 60 minutes. Next, 300 μl of 015M 5M Boutrium buffer (pH 7,6) containing 5% Triton Chemiluminescence was measured in a luminometer (LEADERl, Gen-Probe, C^) by automatic injection of 200111 1 mM HNO, 0,1% H,O, then 2 ooμl of IN NaOH, 2% Z wittergent, followed by 2 seconds of autoinjection. Measured by measuring the signal Ta. For transcription with T7 polymerase, add 5 μm extension mix to 45 μl of transcription mix. In addition to the compound, the final conditions were as follows: 40mM Tris buffer i & (pH 7,9; at 25°C), 6mM MgClv, 2mM spermidium 10mM DTT, 2°5mM ccTP, 2.5sM rUTP, 2.5mM rATP, 6.5mM rGTP and 400 units of T7 RNA polymer. (United 5taLes Bioche+oical Corporation). A 5 μm equivalent aliquot of this sample was subjected to the AE-labeled assay as described above. The oligomers were used for assay. The following results were obtained: Taq Polymer Ze? 4,125 1.083 T7 RNA polymerase 641,753 9 7.218 These data are based on Taq polymerase and T7 RNA polymerase. We show that although sites for enzymes can be created independently of the target region, the activity of these sites is modulated by hybridization to the target. A specific example of the general arrangement shown in FIG. 6E is shown in FIG. 21. This target strand is a synthetic 38a+er oligomer, and the niblob strand forms a four-way connection point with the target strand as shown. Ru. All oligomers were synthesized as described in Example 1 above. Hybridization of the structures shown in Figures 22A-221 (various schematic representations of the structures are shown in Figure 21) The DH analysis properties were evaluated using the DH analysis protocol described in Example 1, and all operations The cultivation was carried out at 50°C. The amount of different chains used is always the same (if not all chains are used) The two target chains are as follows: - 15 pmol; chain 1 - 0.1 pmol; chain 2 - 6.5 pmol; chain 3 - 1.0 pmol. The following results were obtained: Structural addition Hydrolysis (Figure number) Half-life (min) 22A 37.0 22B 0.54 22C1,7 22D 0.72 22E 0.69 22F 0.60 These data demonstrate that the AE is capable of inhibiting ester hydrolysis in the absence of target. Not protected but marked We show that esters are protected from ester hydrolysis in the presence of target (and probe strands). In this embodiment, all four strands must be present to form a stable duplex between the arm regions of probe strands 1 and 2. Target-Independent Probe Amplification In another arrangement, the duplexes formed between complementary arm regions (some of which are formed independently of the target (at least one such duplex is in the presence of (as long as it is formed). Some examples of this arrangement are shown in Figures 19A-19B. One of the utility of this arrangement is that the AE or other label can be placed in multiple regions of a "network" of branched nucleic acid structures. This is amplification.Furthermore, the AE This arrangement could be used for any desired target site if the enables a universal detection complex. Two (or more) probe regions are targeted. Upon hybridization with the target, this structure becomes bound to the target molecule, thus “stabilizing the anchorage linkage point (as shown in Figures 19A and 19B). sea urchin). This bound label amplification network typically has to be separated from the unbound target by some heterogeneous method (e.g., sandwich assay, hydroxyapatite capture of the target nucleic acid). Dew. The number of attachment points formed with the target can be non-limiting (at least 5, or even 1, as long as the hybridized probe does not precipitate from solution). (up to 1000°C, and depending on the size of the probe, can be in the thousands), may bind to the probe before or after hybridization to the target. The same applies to the formation of a new detection network. In one case of the above arrangement, all attachment points could be formed in a target-independent manner. Some examples of this arrangement are shown in Figures 20A-20C. This allows label amplification using branched nucleic acid structures to create networks. However, this would allow the introduction of multiple labels per target site. The probe forms a nucleic acid junction with the target strand (this junction is target dependent in the sense that all junctions cannot form in the absence of the target, but the duplex formed between the binding arm regions target-independent (they form even in the absence of a target), and can use this method with probes that do not form nucleic acid junctions with the target strand. Ru. Therapeutic Probes In addition to detecting and quantifying nucleic acid targets, the branched nucleic acid probe sequences disclosed herein The device can also be used in a variety of therapeutic applications. For example, system 3, the general arrangement shown in Figure 15, makes the probe region specific to, for example, an infectious organism or a particular cancer cell. The arm region is designed to encode a biologically active molecule. It can be used by measuring. This probe connects to the target site. Upon hybridization, the template strand is filled in by an endogenous polymerase (this step may be unnecessary), resulting in an active coding region, which in turn is used to generate biologically active molecules, such as essential transcription or transcription factors. Inhibitors of transduction will produce peptides or other small molecules that are toxic to the organism or cell, or antigens that render the cell susceptible to removal by the immune system (again due to endogenous activity). The arm region may also form double-stranded RNA regions in a target-dependent manner, which in turn stimulates interferon activity. As another example, the target tract (small to form a site that can be cleaved by endogenous lysyl-based enzymes. Connection points formed with the target nucleic acid can be designed. This probe will target the site where this cleavage would be lethal to the organism or cell. As another example, a recognition site for an endogenous nuclease may be created; Rease binds to this recognition site but cleaves at the terminal site on the target mirror; leading to the death of organisms or cells. Additionally, the arm region duplex is an essential endogenous regulatory protein. Recognition sites will be created that compete for protein binding or interfere with the assembly of essential regulatory complexes in a sequence-specific manner. In addition, essential regions of the target nucleic acid Various combinations of probes can be designed to form stable branch points with regions, thus inhibiting the binding of sequence-specific processing factors and would inhibit replication. Other embodiments are within the scope of the following claims. Flgure IA Flgure IB Flgure IC FIGure IE Figure 2D Figure 4A Figure 4B Hybrid 1 Control 1 Flgure 6^ Flgure 6B Half-life (min) +, JLJL 18.19 0.53 34.3 66.52, -IL'L 6 ．． 10 0.70 B, 7 66.33, "L" 22.32 0.55 40. 6 66.14, UL 9.41 0.56 16.8 68J5JUhi 7.67 0.72 10.7 67.86, ``Uhi 8.75' 0.73 12.0 -7, -1L 10 .02 0.56 17.9 67.98, d b 8.44 0.72 11.37 69.39, ff1LIL 8.83 0.75 o, s 64.513, JLJI-7,370,6211,9-- -+4. JIJL 6.76 0.60 11.3 -15, LIL 7.5 1 0.58 12.9 62.216-1LJL 5.79 0.73 7, 9 62.017, Kojidan 5,21 0 .62 8.4 61.618, -Yo Kanhi 6.36 0.64 9.9 61.41-Binding site Special Table Sen7-503139 (22) Flgure 15A Figure 16 TameJ Figure 21 Continuation of front page (51 ) Int, C1,' Identification code Internal reference number Cl2R1:36) (72) Inventor Nelson, Norman Charles United States of America 92111 3639 Maresta Drive I, San Diego, California (72) Inventors Betsverkov, Robert United States 92007 California 950 Norbay Street, Cardiff Pie the Sea, Near

Claims (74)

[Claims] 1. A method for detecting the presence or amount of a target nucleic acid in a sample, the method comprising: The method consists of the following steps: one or more nucleic acid molecules with said sample and when said target nucleic acid is present. The molecules are brought into contact under predetermined ambient conditions that allow hybridization. (wherein the one or more nucleic acid molecules are isolated under the predetermined ambient conditions) at least two independent target regions that hybridize with the target nucleic acid; with said target nucleic acid under predetermined ambient conditions or with each other in the absence of said target nucleic acid. but not hybridize to each other in the presence of the target nucleic acid. at least two arm regions); and any hybrids of said arm regions. Dyzation is detected as an indication of the presence or amount of the target nucleic acid. 2. The detection step includes detecting one or more nucleic acid molecules and the target nucleic acid in resolvase. and detecting cleavage of the one or more nucleic acid molecules by the resolbase. 2. The method of claim 1, comprising: 3. The detection step performs DNA footprint analysis of one or more nucleic acid molecules. 2. The method according to claim 1, comprising: 4. The detection step determines the mobility of one or more nucleic acid molecules within the gel matrix. 2. A method according to claim 1, comprising observing. 5. The detection step involves binding of the intercalary to one or more nucleic acid molecules. 2. The method according to claim 1, comprising detecting a match. 6. the detecting step contacts the one or more nucleic acid molecules with an S1 nuclease; detecting any cleavage of the one or more nucleic acid molecules by the nuclease; 2. The method of claim 1, comprising: 7. The detection step contacts the one or more nucleic acid molecules with a restriction endonuclease. and detecting cleavage of the one or more nucleic acid molecules by the endonuclease. 2. The method of claim 1, comprising: 8. The detection step may involve measuring the thermal stability of one or more nucleic acid molecules. 2. The method according to claim 1. 9. One or more nucleic acid molecules may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. susceptible to chemical modification by acids or bases when forming part of the molecule; This occurs when the molecule is present within the single-stranded nucleic acid molecule or other double-stranded nucleic acid molecule. containing an insertion molecule that is not sensitive to chemical modification such as Contacting the above nucleic acid molecule with the chemical substance and measuring the amount of chemical modification of the molecule. 2. The method of claim 1, comprising: 10. 10. The method of claim 9, wherein the intercalating agent is an acridinium ester. 11. At least one of the 1 or more nucleic acid molecules is 1000 neighbors of the target nucleic acid 2. The target region according to claim 1, comprising a target region comprising 8 to 100 contiguous bases complementary to the region of the contact base. How to put it on. 12. At least one of the one or more nucleic acid molecules is a 50-contiguous salt of the target nucleic acid. The method according to claim 1, comprising a target region comprising 8 to 30 contiguous bases complementary to the region of the group. Law. 13. The arm region is at the hybridization temperature under predetermined ambient conditions. Claim 1, wherein a duplex having a melting temperature 4°C lower is formed in the absence of a target nucleic acid. Method described. 14. The arm region is at a hybridization temperature under predetermined ambient conditions. form a duplex in the absence of a target nucleic acid that has a melting temperature at least 7°C lower than The method according to claim 1. 15. The arm region is at the hybridization temperature under predetermined ambient conditions. forming a duplex in the absence of a target nucleic acid with a melting temperature at least 10°C lower than 2. The method according to claim 1. 16. The arm region is at a hybridization temperature under predetermined ambient conditions. Claim 1, wherein a duplex having a melting temperature 4°C higher is formed in the presence of the target nucleic acid. Method described. 17. The arm region is at a hybridization temperature under predetermined ambient conditions. Claim 1, wherein a duplex having a melting temperature 7°C higher is formed in the presence of the target nucleic acid. Method described. 18. The arm region is at a hybridization temperature under predetermined ambient conditions. Claim 1 wherein a duplex having a melting temperature 10°C higher is formed in the presence of the target nucleic acid. The method described in. 19. a unique nucleic acid molecule is provided, the nucleic acid molecule having at least two arm regions; 2. The method of claim 1, comprising a binding loop region. 20. One or more nucleic acid molecules each containing a target region and an arm region 2. The method of claim 1, comprising two nucleic acid molecules. 21. The one or more nucleic acid molecules each contain at least one arm region. consisting of three nucleic acid molecules, at least two of which have independent target regions. between the arm regions, and the three nucleic acid molecules hybridize with the target nucleic acid to form a region between the arm regions. 2. The method of claim 1, wherein at least two independent duplexes are formed. 22. The one or more nucleic acid molecules each contain at least one arm region. consisting of four nucleic acid molecules, at least two of which have independent target regions. The four nucleic acid molecules and the target nucleic acid hybridize to form a region between the arm regions. 2. The method of claim 1, wherein at least three independent duplexes are formed. 23. The one or more nucleic acid molecules each contain at least one arm region. consisting of five nucleic acid molecules, at least two of which have independent target regions. The five nucleic acid molecules and the target nucleic acid hybridize to form a region between the arm regions. 2. The method of claim 1, wherein at least four independent duplexes are formed. 24. The target region hybridizes with the target nucleic acid, and the arm regions hybridize together. to form an arm and a connection point at the base of the arm between two separate target areas. is formed, One or more nucleic acid molecules or said target nucleic acids are located near said point of attachment in said arm region. region, does not form a duplex with the target region or the target nucleic acid, and loops outward from the connection point. or the target region may contain a nucleic acid that makes a At the end remote from the arm region, the target nucleic acid is bonded to the target nucleic acid without forming a double strand with the target nucleic acid. loops from the double strands formed between the target regions or as single-stranded regions. comprising a nucleic acid that either extends from the end of the target region; or The arm region extends from the arm region without forming a duplex with other arm regions. the arm region forming a loop or separating from the target region as a single-stranded molecule; 2. The method of claim 1, comprising a nucleic acid extending from the end of. 25. The target region hybridizes to the target nucleic acid, and the arm regions hybridize together. to form an arm, with a connection point at the base of the arm between two separate target areas. formed, and one said arm region is capable of hybridizing to another arm region. used for duplex formation with another nucleic acid molecule to form a second arm 2. The method of claim 1, further comprising a possible single-stranded region at the end furthest from the target region. Method. 26. 26. The method of claim 25, wherein both arm regions include single stranded regions. 27. One or more nucleic acid molecules can form a duplex with a single-stranded region to form a second or more forms a third arm and a second connection point between the two or more such arms; 27. The method of claim 26, comprising the nucleic acid molecule obtained. 28. At least two arm regions are activated when hybridized with a target nucleic acid. 2. A biologically or chemically active site according to claim 1. the method of. 29. 29. The biologically active site is a restriction endonuclease site. the method of. 30. the biologically active site comprises a double-stranded region and a single-stranded region, the double-stranded region 29. The method according to claim 28, wherein the method acts as a primer for a DNA polymerase. . 31. Claim that the biologically active site comprises a promoter for RNA polymerase The method according to item 28. 32. A promoter can be transcribed to form multiple RNA transcripts. The method according to claim 31. 33. DNA/R whose biologically active site is susceptible to cleavage by RNAseH 29. The method of claim 28, comprising an NA duplex. 34. A claim containing a chemical substance capable of cleaving adjacent double-stranded nucleic acids with chemically active sites 28. The method described in 28. 35. 35. The method of claim 34, wherein the chemical comprises Fe.EDTA. 36. 35. The method of claim 34, wherein the chemical comprises phenanthroline. 37. One or more nucleic acid molecules and a target nucleic acid and an arm region or a single-stranded region extending from the arm region to form one or more other arms; Claim 1, further comprising contacting another nucleic acid molecule capable of hybridizing. the method of. 38. Other nucleic acid molecules hybridize between them to create numerous other arm regions. 38. The method of claim 37, wherein 39. cleavage of the site with a substance that reduces the stability of the target region and the target nucleic acid; This causes one or more other nucleic acid molecules to hybridize to the target nucleic acid. 29. The method of claim 28, wherein the method is capable of allowing 40. Claim 1 wherein one of the one or more nucleic acid molecules comprises a phosphorothioate. The method described in. 41. One or more nucleic acid molecules hybridize to a complementary nucleic acid molecule a single-stranded nucleic acid capable of forming a triple-stranded nucleic acid molecule with one arm region or target region 2. The method of claim 1, comprising a region. 42. at least two that hybridize with the target nucleic acid under predetermined ambient conditions. two independent target regions and said target nucleic acid under said predetermined ambient conditions; or do not hybridize with each other in the absence of the target nucleic acid, but in the presence of the target nucleic acid. 1 or 2 together comprising at least two arm regions that hybridize with each other A purified nucleic acid probe consisting of the above nucleic acid molecules. 43. One or more nucleic acid molecules may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule. susceptible to chemical modification by acids or bases when forming part of an acid molecule , when said molecule is present in said single-stranded nucleic acid molecule or other in said double-stranded nucleic acid molecule. 43. The probe of claim 42, comprising an insensitive insertion molecule. 44. 44. The probe of claim 43, wherein the intercalant is an acridinium ester. 45. At least one of the 1 or more nucleic acid molecules is 50 contiguous bases of the target nucleic acid Claim 42, comprising a target region comprising 8 to 30 complementary bases capable of complexing with a region of Probe as described. 46. At least one of the 1 or more nucleic acid molecules is 1000 contiguous to the target nucleic acid A claim comprising a target region containing 8 to 100 complementary bases that can be complexed with a region of bases. 42. 47. The arm region is at the hybridization temperature under predetermined ambient conditions. 43. A duplex having a Tm lower than 4° C. is formed in the absence of a target nucleic acid. Probe on board. 48. The arm region is at the hybridization temperature under predetermined ambient conditions. A claim that forms a duplex in the absence of a target nucleic acid with a Tm that is at least 7°C lower than Probe according to item 42. 49. The arm region is at the hybridization temperature under predetermined ambient conditions. 43. A duplex having a Tm lower than 10° C. is formed in the absence of a target nucleic acid. Probe as described. 50. The arm region is at the hybridization temperature under predetermined ambient conditions. 43. A duplex having a Tm higher than 4° C. is formed in the presence of the target nucleic acid. Probe on board. 51. The arm region is at a hybridization temperature under predetermined ambient conditions. 43. A duplex having a Tm higher than 7° C. is formed in the presence of the target nucleic acid. Probe on board. 52. The arm region is at the hybridization temperature under predetermined ambient conditions. 43. A duplex having a Tm higher than 10° C. is formed in the presence of the target nucleic acid. Probe as described. 53. a unique nucleic acid molecule is provided, the nucleic acid molecule having at least two arm regions; 43. The probe of claim 42, comprising a binding loop region. 54. One or more nucleic acid molecules each containing a target region and an arm region 43. The probe according to claim 42, comprising two nucleic acid molecules comprising: 55. The one or more nucleic acid molecules each contain at least one arm region. consisting of three nucleic acid molecules, at least two of which have independent target regions. The three nucleic acid molecules hybridize with the target nucleic acid to generate a small number of arm regions. 43. The probe of claim 42, which forms at least two independent hybridized pairs. . 56. One or more nucleic acid molecules each comprising at least one arm region consisting of four nucleic acid molecules, at least two of which have independent target regions. The four nucleic acid molecules and the target nucleic acid hybridize to form a link between the arm regions. 43. The probe of claim 42, which forms at least three independent duplexes. 57. The one or more nucleic acid molecules each contain at least one arm region. consisting of five nucleic acid molecules, at least two of which have independent target regions. The five nucleic acid molecules and the target nucleic acid hybridize to form a link between the arm regions. 43. The method of claim 42, wherein at least four independent duplexes are formed. 58. The target region hybridizes with the target nucleic acid, and the arm regions hybridize together. to form an arm and a connection point at the base of the arm between two separate target areas. is formed, One or more nucleic acid molecules or said target nucleic acids are located near said point of attachment in said arm region. region, does not form a duplex with the target region or the target nucleic acid, and loops outward from the connection point. or the target region may contain a nucleic acid that makes a At the end remote from the arm region, there is a target nucleic acid that does not form a double strand with the target nucleic acid. loops from the double strands formed between the target regions or as single-stranded regions. comprising a nucleic acid that either extends from the end of the target region; or The arm region extends from the arm region without forming a duplex with other arm regions. the arm region forming a loop or separating from the target region as a single-stranded molecule; 43. The probe of claim 42, comprising a nucleic acid extending from the end of. 59. The target region hybridizes with the target nucleic acid, and the arm regions hybridize together. to form an arm, with a connection point at the base of the arm between two separate target areas. formed, and one said arm region is capable of hybridizing to another arm region. used for duplex formation with another nucleic acid molecule to form a second arm 43. Further comprising a possible monolithic region at the end furthest from the target region. probe. 60. 60. The probe of claim 59, wherein both arm regions include a single region. 61. One or more nucleic acid molecules form a duplex with a single-stranded region to form a second or comprises a third arm and a nucleic acid molecule capable of forming a second point of attachment between the arms. 61. A probe according to claim 60. 62. At least two arm regions are activated when hybridized with a target nucleic acid. 43. The biologically active site of claim 42, wherein the arm comprises a biologically or chemically active site. Probe on board. 63. 63. The protein of claim 62, wherein the biological activity is a restriction endonuclease site. robe. 64. the biologically active site comprises a double-stranded region and a single-stranded region, the double-stranded region 63. The process according to claim 62, which acts as a primer for a DNA polymerase. -bu. 65. Claim that the biologically active site comprises a promoter for RNA polymerase Probe according to item 62. 66. A promoter can be transcribed to form multiple RNA transcripts. 66. The probe according to claim 65. 67. DNA/R whose biologically active site is susceptible to cleavage by RNAseH 63. The probe of claim 62, comprising an NA duplex. 68. A claim containing a chemical substance capable of cleaving a double-stranded nucleic acid in close proximity to a chemically active site 62. 69. 69. The probe of claim 68, wherein the chemical comprises Fe.EDTA. 70. 69. The probe of claim 68, wherein the chemical comprises phenanthroline. 71. an arm region or one or more arms extending from the arm region; A claim further comprising other nucleic acid molecules capable of hybridizing with the single-stranded region forming the 42. 72. Other nucleic acid molecules hybridize between them to form multiple arm regions 72. The probe of claim 71, which can be 73. Claim 4 wherein one of the one or more nucleic acid molecules comprises a phosphorothioate. 2. The probe according to 2. 74. One or more nucleic acid molecules hybridize to a complementary nucleic acid molecule a single-stranded nucleic acid capable of forming a triple-stranded nucleic acid molecule with one arm region or target region 43. The probe of claim 42, comprising a region.